Method for preparing acylated crosslinked glycosaminoglycans

Abstract

A method of preparing a hydrogel product including crosslinked glycosaminoglycan molecules, said method including: i) providing a glycosaminoglycan crosslinked by amide bonds, wherein the crosslinked glycosaminoglycans include residual amine groups; and ii) acylating residual amine groups of the crosslinked glycosaminoglycans provided in i) to form acylated crosslinked glycosaminoglycans.

The invention claimed is: 
     
       1. A hydrogel comprising acylated crosslinked glycosaminoglycans (GAGs), wherein the crosslinked GAGs comprise:
 an at least partially deacetylated GAG comprising free amine groups; and 
 a second GAG, 
 wherein the at least partially deacetylated GAG is crosslinked to the second GAG by amide bonds formed between 2′-amino groups of the at least partially deacetylated GAG and activated carboxyl groups of the second GAG; and 
 wherein the hydrogel has residual free amine groups and a degree of acetylation of at least 95%. 
 
     
     
       2. The hydrogel according to  claim 1 , wherein the crosslinked GAGs are prepared by a method comprising:
 (a) obtaining a solution comprising the at least partially deacetylated GAG, and the second GAG; 
 (b) activating carboxyl groups on the at least partially deacetylated GAG and/or the second GAG with a coupling agent, forming activated GAGs; 
 (c) crosslinking the activated GAGs via their activated carboxyl groups using 2′-amino groups of the at least partially deacetylated GAGs, forming the amide bond-crosslinked GAGs. 
 
     
     
       3. The hydrogel according to  claim 2 , wherein the at least partially deacetylated GAG is selected from the group consisting of deacetylated hyaluronic acid, deacetylated chondroitin, and deacetylated chondroitin sulfate, and mixtures thereof. 
     
     
       4. The hydrogel according to  claim 2 , wherein the at least partially deacetylated GAG is deacetylated hyaluronic acid. 
     
     
       5. The hydrogel according to  claim 2 , wherein the at least partially deacetylated GAG has a degree of acetylation of 95% or less, and a weight average molecular weight of 0.1 MDa or more. 
     
     
       6. The hydrogel according to  claim 2 , wherein the at least partially deacetylated glycosaminoglycan is prepared by:
 (a) allowing a GAG comprising N-acetyl groups to react with hydroxylamine (NH 2 OH), or a salt thereof, at a temperature of 100° C. or less for 2-200 hours to form an at least partially deacetylated GAG, and 
 (b) recovering the at least partially deacetylated GAG. 
 
     
     
       7. The hydrogel according to  claim 2 , wherein the second GAG is selected from the group consisting of hyaluronic acid, chondroitin, and chondroitin sulfate, and mixtures thereof. 
     
     
       8. The hydrogel according to  claim 2 , wherein the second GAG is hyaluronic acid. 
     
     
       9. The hydrogel according to  claim 2 , wherein the coupling agent is a peptide coupling reagent. 
     
     
       10. The hydrogel according to  claim 2 , wherein the coupling reagent is DMTMM. 
     
     
       11. The hydrogel according to  claim 2 , wherein the method of preparing the crosslinked GAGs further comprises acetylating residual free amine groups of the crosslinked GAGs to form acetylated crosslinked GAGs. 
     
     
       12. The hydrogel according to  claim 2 , wherein the method of preparing the crosslinked GAGs further comprises subjecting the crosslinked GAGs to an alkaline solution to hydrolyze ester crosslinks formed as byproducts during the amide crosslinking. 
     
     
       13. The hydrogel according to  claim 12 , wherein the alkaline solution comprises sodium hydroxide or potassium hydroxide. 
     
     
       14. The hydrogel according to  claim 13 , wherein the crosslinked GAGs are subjected to the sodium hydroxide solution at a pH between 12-14 for 0.5 to 4 hours. 
     
     
       15. The hydrogel according to  claim 1 , wherein the hydrogel is in a solid dry form. 
     
     
       16. The hydrogel according to  claim 1 , wherein the hydrogel is precipitated in an alcohol. 
     
     
       17. A composition comprising:
 the hydrogel according to  claim 1 ; and 
 water. 
 
     
     
       18. The composition according to  claim 17 , wherein the composition is formulated as an injectable composition. 
     
     
       19. The composition according to  claim 17 , wherein the hydrogel is suspended in saline solution. 
     
     
       20. The composition according to  claim 19 , wherein the pH of the saline solution is 7.4. 
     
     
       21. The hydrogel according to  claim 1 , wherein the crosslinked GAGs are substantially free of ester crosslinks. 
     
     
       22. The hydrogel according to  claim 1 , wherein the crosslinked GAGs comprise two different GAGs selected from hyaluronic acid, chondroitin, chondroitin sulphate, heparin sulphate, heparosan, heparin, dermatan sulphate, and keratin sulphate. 
     
     
       23. The hydrogel according to  claim 1 , wherein the second GAG is non-deacetylated.